The homeostasis of endocrine and neuroendocrine systems relies on the ability of cells to correctly sort, process and package cargo molecules into secretory granules. The goal of this project is a determination of the factors that regulate protein sorting in neuroendocrine cells and maintain the organelle-specific microenvironments. One approach to address how neuroendocrine cells compartmentalize biochemical reactions has been to study the biochemistry and cell biology of the proprotein convertases (PCS), which catalyze the proteolytic activation of peptide hormones and bioactive proteins in distinct secretory pathway compartments. For example, furin, a ubiquitously expressed, type-I membrane PC, is localized to the trans- Golgi network (TGN) and processes many bioactive proteins in both the biosynthetic and endocytic pathways. By contrast, PC1/3 and PC2 reside in the regulated secretory pathway and process prohormone molecules. The PCS are synthesized as proenzymes that require autoproteolytic activation. Activation of furin in vitro requires an ordered series of cleavages within its autoinhibitory propeptide that are pH- and calcium- dependent and reflect the gradient of these factors that exists between the endoplasmic reticulum and the TGN. Because other PCS including PC1/3 and PC2 contain similarly organized propeptides, the furin activation pathway may serve as a model for other members of this family. In specific aim 1, the in vivo furin activation pathway will be determined and compared to the activation pathway of PC1/3. The trafficking of the PCS reveals a biochemical basis for regulated pathway sorting. Furin, PC1/3 and PC2 bud from he TGN into immature secretory granules (ISGs). Whereas PC1/3 and PC2 are then transferred to mature secretory granules (MSGs), furin is not. Exclusion of furin from MSGs requires the phosphorylation of its cytosolic domain (cd) at a casein kinase II (CKII) site. These results suggest a CKII-dependent local cycling of furin between the TGN and ISGs. In specific aim 2, the hypothesis that furin is retrieved from ISGs and returned to the TGN will be tested, including the role of phosphorylation. In specific aim 3, the hypothesis that CKII is the in vivo furin kinase will be examined. Finally, the phosphorylation-dependent retrieval of furin from the regulated pathway argues for the presence of a phosphorylated furin-cd binding protein that directs the trafficking of the endoprotease. In specific aim 4, the role of the first identified phosphorylation-dependent furin-cd binding protein, 45.1.1, in the trafficking of furin in neuroendocrine cells will be determined.